Teramoto N, Szekely L, Pokrovskaja K, Hu L F, Yoshino T, Akagi T, Klein G
Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden.
J Virol Methods. 1998 Jul;73(1):89-97. doi: 10.1016/s0166-0934(98)00048-2.
Simultaneous detection of two antigens by immunostaining usually requires primary antibodies from two different species or a hapten modification of one of the antibodies if they are from the same species. A novel double staining method is described for immunodetection of two independent antigens using two mouse monoclonal antibodies. The principle of the method is the following: The first antigen is detected by a monoclonal antibody that is diluted so extensively that it cannot be recognized with conventional detection systems. A highly sensitive biotin-tyramide amplification system is used to identify this antibody. The second antigen is stained with a monoclonal antibody by dilution and detected by conventional immunostaining. The method was tested for both alkaline-phosphatase staining on paraffin sections and immunofluorescence staining on cultured cells in cytospin preparation. The absence of cross-reaction in the former system was demonstrated by the mutually exclusive detection of T- and B-cells in human lymph nodes or T-cells and carcinoma cells in nasopharyngeal carcinoma biopsies. Similarly, the EBV encoded EBNA2 and ZEBRA proteins showed a mutually exclusive staining by immunofluorescence on B95-8 cells. The method could be used to demonstrate co-expression of two independent antigens in the same cells, such as PCNA and keratin in carcinoma cells in paraffin sections and for EBNA2 and LMP1 EBV proteins in immunofluorescence preparations of B95-8 cells.
通过免疫染色同时检测两种抗原通常需要来自两种不同物种的一抗，或者如果它们来自同一物种，则需要对其中一种抗体进行半抗原修饰。本文描述了一种使用两种小鼠单克隆抗体免疫检测两种独立抗原的新型双重染色方法。该方法的原理如下：第一种抗原由一种单克隆抗体检测，该抗体稀释程度极高，以至于传统检测系统无法识别。使用高灵敏度的生物素-酪胺扩增系统来识别这种抗体。第二种抗原用单克隆抗体通过稀释进行染色，并通过传统免疫染色进行检测。该方法在石蜡切片的碱性磷酸酶染色和细胞涂片制备的培养细胞免疫荧光染色中均进行了测试。在前一种系统中，通过在人淋巴结中对T细胞和B细胞的相互排斥检测，或在鼻咽癌活检中对T细胞和癌细胞的相互排斥检测，证明了不存在交叉反应。同样，EBV编码的EBNA2和ZEBRA蛋白在B95-8细胞上通过免疫荧光显示出相互排斥的染色。该方法可用于证明两种独立抗原在同一细胞中的共表达，如石蜡切片中癌细胞中的PCNA和角蛋白，以及B95-8细胞免疫荧光制剂中的EBNA2和LMP1 EBV蛋白。
